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Editorials |
1 Department of Clinical Chemistry, Georg-August University, Robert Koch Strasse 40, 37075 Goettingen, Germany, Fax 49-551-39-12504, E-mail nahsen{at}gwdg.de
The advent of the PCR has revolutionized many molecular biological techniques, including single-nucleotide polymorphism (SNP) detection. Interest in the detection and discovery of human polymorphisms increased further with the complete sequencing of the human genome. The main applications for SNP detection include diagnosis of genetic disease and identification of functional mutations in genes that are of pharmacogenetic importance. The results from testing for somatic mutations remain with a person for a lifetime, and retesting is performed only rarely. To be able to deliver a molecular diagnosis of the highest quality, we must have a sound understanding of the different principles used for the detection of polymorphic DNA.
The clinical laboratory today is equipped with a plethora of assays for the detection of SNPs, and new detection methods continue to be published. Molecular diagnostic assays often make use of the real-time monitoring of hybridization for the detection of base mutations. A common assay set-up uses a fluorescence resonance energy transfer (FRET) probe combination. A 3'-dye-labeled detection probe together with a 5'-dye-labeled and 3'-phosphorylated anchor probe interact by FRET in the hybridized state. In the unhybridized state, no FRET occurs, making this technique applicable to homogeneous single-tube assays. Variations of this theme include the use of exonuclease probes and molecular beacons. Initially it was recommended to avoid attaching the dye to a terminal guanosine residue because quenching was observed (1), but for molecular beacons, a design modification was suggested. Instead of the second dye or quencher, a string of guanosine bases was opposed to the single dye on the stem structure of the beacon (2). This simple measure provided sufficient quenching, made probe chemistry cheaper and easier, and decreased background fluorescence.
The effects of neighboring nucleobases on dye fluorescence emission have been known for a long time [see Ref. (3) and references therein], but only recently have these effects been used for real-time PCR (4)(5). A common observation was that a G base must be proximate to the dye for sufficient quenching, a finding that is in line with results from physical chemistry (3). To date, however, only the melting transition of labeled oligonucleotide probes has been observed, for example, when Crockett and Wittwer set up different SNP detection assays using only a single fluorescein-labeled detection probe in the assay (4). In this issue of Clinical Chemistry, Gundry et al. (6) show how the melting behavior of a DNA strand generated by PCR with a dye-labeled primer is altered by the presence of a base alteration. The assay is straightforward and has potential for both genotyping and mutation scanning. The PCR product is amplified, melted, and reannealed to generate a mixture of homo- and heteroduplex DNA, which is melted in an analytical melting cycle. Alterations from the melting transition of a reference sequence included in the run indicate the presence of a mutation. The advantages are the simple probe chemistry and the homogeneous method.
Where does this method have potential? In the genotyping of known mutations, in the search for unknown mutations (scanning), or both? It can be assumed that the more sensitive a method is for the one, the less sensitive it will be for the other. The use of hybridization probes comes close to a reference method for genotyping, especially if the wild-type and mutant sequences are each probed individually. Only problems inherent to PCR itself, such as impaired primer binding because of an unexpected mutation, could still cause genotyping errors. This is a long-known source of error (7), but it is difficult to control for this kind of assay. The molecular basis of hybridization assays is disturbed probe binding because of mismatches. The stability of oligonucleotide DNA can be calculated with a thermodynamic nearest-neighbor model (8). This puts probe-based genotyping on solid ground (9). However, the method is limited to short regions of a known sequence.
The melting transitions of longer strands (polymeric DNA) are also altered by the presence of a SNP. It is easily understood that if the destabilization of a carefully chosen 20mer detection probe by a mismatch is in the range of 7–10 °C, that of a longer strand will be much less. If these small differences could be detected reliably, it would be an elegant method for mutation scanning. A melting study reported a 0.6–3.8 °C decrease in the melting temperature in the presence of the SNP when PCR products of 100–167 bp containing various SNPs were used (10). Although this may be acceptable for mutation scanning studies, it is not well suited for a routine genotyping assay because minor assay variations could cause incorrect genotyping results (11). The melting transition of polymeric DNAs cannot be described by a nearest-neighbor model. They typically melt cooperatively in different domains and in several intermediate subtransitions (12). The calculation of these melting domains needs a statistical model such as the Poland algorithm (13).
The method presented in this issue by Gundry et al. (6) shows characteristics of both probe-based genotyping, characterized by favorable detection of homozygote mutations, and strand melting-based mutation scanning, characterized by sensitivity toward different mutations over (potentially) one of the strand’s melting domains. For their functional assays, Gundry et al. attached the dye to terminal guanosines and, in one case, to a terminal thymidine, but then the two neighboring bases were G and C. This is in line with another systematic study that showed that fluorescence increase on duplex formation requires at least one guanosine within 4 bp on either side of the label (14). This design criterion poses almost no constraints on primer choice. A minor point may be effects from the terminal transferase activity of Taq polymerase that often adds a 3' nontemplate adenosine to the PCR-amplified product. This base is also expected to interact with the dye, but its addition can be avoided by the choice of other PCR enzymes.
The effects of nucleobase quenching on commonly used dyes have been studied in detail. The findings of Marras et al. (15) confirm an earlier report that guanosine is the best quencher (3). Sensitive dyes are, for example, 6-carboxyfluorescein (FAM) and TET, whereas Cy5 and Cy5.5 are probably not good choices (15). It is not yet clear which domains are really informative, i.e., in which domain a SNP can be detected. The authors speculate that it is the domain that contains the labeled primers. The assumption makes sense intuitively but is not supported by calculations with the Poland algorithm (see the online supplemental data accompanying the article). The mutation-induced fluorescence change may be caused by secondary structure or melting domain alterations that are not adequately accounted for in the algorithm. In fact, mutation detection with labeled primers may or may not work for mutations in certain domains. This puts the assay on uncertain ground in this matter. Further studies are needed to fully understand which domains can be scanned with respect to the position of SNPs within melting domains and their distance to the labeled primer.
Knowledge of the underlying thermodynamics of base-pairing and nucleobase dye interactions allows a better understanding and more rational design of oligonucleotide-based assays. The previously intuition-guided art of oligonucleotide sequence selection should now be replaced by a more scientific approach.
The report by Gundry et al. (6) describes a novel, simple homogeneous assay to detect SNPs. This procedure requires only a single labeled primer and one unlabeled primer, as opposed to conventional FRET-based methods that need two primers and two labeled probes. Furthermore, the new procedure has the potential for detecting previously unknown mutations within the amplicon.
References
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